Cellular Functions of High-Temperature Requirement Factor A4 in Placenta.

The expression of High-temperature requirement factor A4 (HtrA4) mRNA is significantly lower in the chorionic villi of patients with recurrent pregnancy loss (RPL) than in the control group. We conducted an investigation into the cellular functions of HtrA4 using the CRISPR/Cas9 system and shRNA-HtrA4 to create knockout BeWo cells and HtrA4 knockdown JEG3 cells. Our results indicated that the knockout BeWo cells exhibited reduced capacity for invasion and fusion, but increased levels of proliferation and migration, with a significantly shortened cell cycle compared to wild-type cells. Wild-type BeWo cells highly expressed cell invasion- and fusion-related factors, while knockout BeWo cells highly expressed migration-, proliferation-, and cell cycle-related factors. The shRNA-HtrA4 JEG3 cells showed a decreased capacity for invasion, but an increased capacity for migration, accompanied by a decrease in the expression of cell invasion-related factors and an increase in migration-related factors. Moreover, our ELISA results revealed that the serum HtrA4 level was lower in patients with RPL than in the controls. These findings suggest that HtrA4 depletion may be associated with placental dysfunction.

Two Arabidopsis Splicing Factors, U2AF65a and U2AF65b, Differentially Control Flowering Time by Modulating the Expression or Alternative Splicing of a Subset of FLC Upstream Regulators.

We investigated the transcriptomic changes in the shoot apices during floral transition in Arabidopsis mutants of two closely related splicing factors: AtU2AF65a (atu2af65a) and AtU2AF65b (atu2af65b). The atu2af65a mutants exhibited delayed flowering, while the atu2af65b mutants showed accelerated flowering. The underlying gene regulatory mechanism of these phenotypes was unclear. We performed RNA-seq analysis using shoot apices instead of whole seedlings and found that the atu2af65a mutants had more differentially expressed genes than the atu2af65b mutants when they were compared to wild type. The only flowering time gene that was significantly up- or down-regulated by more than two-fold in the mutants were FLOWERING LOCUS C (FLC), a major floral repressor. We also examined the expression and alternative splicing (AS) patterns of several FLC upstream regulators, such as COOLAIR, EDM2, FRIGIDA, and PP2A-b'ɤ, and found that those of COOLAIR, EDM2, and PP2A-b'ɤ were altered in the mutants. Furthermore, we demonstrated that AtU2AF65a and AtU2AF65b genes partially influenced FLC expression by analyzing these mutants in the flc-3 mutant background. Our findings indicate that AtU2AF65a and AtU2AF65b splicing factors modulate FLC expression by affecting the expression or AS patterns of a subset of FLC upstream regulators in the shoot apex, leading to different flowering phenotypes.

Correction: Kang et al. Preparation and In Vivo Evaluation of a Lidocaine Self-Nanoemulsifying Ointment with Glycerol Monostearate for Local Delivery. Pharmaceutics 2021, 13, 1468.

The authors wish to make the following corrections to this paper [...].

Preparation and In Vivo Evaluation of a Lidocaine Self-Nanoemulsifying Ointment with Glycerol Monostearate for Local Delivery.

Lidocaine, a commonly used local anesthetic, has recently been developed into a number of ointment products to treat hemorrhoids. This study examined its efficient delivery to the dermis through the pharmaceutical improvement of hemorrhoid treatment ointments. We attempted to increase the amount of skin deposition of lidocaine by forming a nanoemulsion through the self-nanoemulsifying effect that occurs when glycerol monostearate (GMS) is saturated with water. Using Raman mapping, the depth of penetration of lidocaine was visualized and confirmed, and the local anesthetic effect was evaluated via an in vivo tail-flick test. Evaluation of the physicochemical properties confirmed that lidocaine was amorphous and evenly dispersed in the ointment. The in vitro dissolution test confirmed that the nanoemulsifying effect of GMS accelerated the release of the drug from the ointment. At a specific concentration of GMS, lidocaine penetrated deeper into the dermis; the in vitro permeation test showed similar results. When compared with reference product A in the tail-flick test, the L5 and L6 compounds containing GMS had a significantly higher anesthetic effect. Altogether, the self-nanoemulsifying effect of GMS accelerated the release of lidocaine from the ointment. The compound with 5% GMS, the lowest concentration that saturated the dermis, was deemed most appropriate.

Arabidopsis U2AF65 Regulates Flowering Time and the Growth of Pollen Tubes.

During pre-mRNA splicing, U2 small nuclear ribonucleoprotein auxiliary factor 65 (U2AF65) interacts with U2AF35 and splicing factor 1 (SF1), allowing for the recognition of the 3'-splice site by the ternary complex. The functional characterization of U2AF65 homologs has not been performed in Arabidopsis thaliana yet. Here, we show that normal plant development, including floral transition, and male gametophyte development, requires two Arabidopsis U2AF65 isoforms (AtU2AF65a and AtU2AF65b). Loss-of-function mutants of these two isoforms displayed opposite flowering phenotypes: atu2af65a mutants showed late flowering, whereas atu2af65b mutants were characterized by slightly early flowering, as compared to that in the wild-type (Col-0) plants. These abnormal flowering phenotypes were well-correlated with the expression patterns of the flowering time genes such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). However, the two atu2af65 mutants did not display any morphological abnormalities or alterations in abiotic stress tests. Double mutation of the AtU2AF65a and AtU2AF65b genes resulted in non-viable seeds due to defective male gametophyte. In vitro pollen germination test revealed that mutations in both AtU2AF65a and AtU2AF65b genes significantly impaired pollen tube growth. Collectively, our findings suggest that two protein isoforms of AtU2AF65 are differentially involved in regulating flowering time and display a redundant role in pollen tube growth.

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms.

KEY MESSAGE: The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes.

KEY MESSAGE: The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-ß transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

OsNF-YC2 and OsNF-YC4 proteins inhibit flowering under long-day conditions in rice.

MAIN CONCLUSION: OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes ( Ehd1, Hd3a , and RFT1 ) in rice. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors control numerous developmental processes by forming heterotrimeric complexes, but little is known about their roles in flowering in rice. In this study, it is shown that some subunits of OsNF-YB and OsNF-YC interact with each other, and among them, OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response of rice. Protein interaction studies showed that the physical interactions occurred between the three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11). Repression and overexpression of the OsNF-YC2 and OsNF-YC4 genes revealed that they act as inhibitors of flowering only under long-day (LD) conditions. Overexpression of OsNF-YC6, however, promoted flowering only under LD conditions, suggesting it could function as a flowering promoter. These phenotypes correlated with the changes in the expression of three rice flowering-time genes [Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1)]. The diurnal and tissue-specific expression patterns of the subsets of OsNF-YB and OsNF-YC genes were similar to those of CCT domain encoding genes such as OsCO3, Heading date 1 (Hd1) and Ghd7. We propose that OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response by interacting directly with OsNF-YB8, OsNF-YB10 or OsNF-YB11 proteins in rice.

Efficacy of corifollitropin alfa followed by recombinant follicle-stimulating hormone in a gonadotropin-releasing hormone antagonist protocol for Korean women undergoing assisted reproduction.

OBJECTIVE: To evaluate the effect of a gonadotropin-releasing hormone (GnRH) antagonist protocol using corifollitropin alfa in women undergoing assisted reproduction. METHODS: Six hundred and eighty-six in vitro fertilization-embryo transfer (IVF)/intracytoplasmic sperm injection (ICSI) cycles were analyzed. In 113 cycles, folliculogenesis was induced with corifollitropin alfa and recombinant follicle stimulating hormone (rFSH), and premature luteinizing hormone (LH) surges were prevented with a GnRH antagonist. In the control group (573 cycles), premature LH surges were prevented with GnRH agonist injection from the midluteal phase of the preceding cycle, and ovarian stimulation was started with rFSH. The treatment duration, quality of oocytes and embryos, number of embryo transfer (ET) cancelled cycles, risk of ovarian hyperstimulation syndrome (OHSS), and the chemical pregnancy rate were evaluated in the two ovarian stimulation protocols. RESULTS: There were no significant differences in age and infertility factors between treatment groups. The treatment duration was shorter in the corifollitropin alfa group than in the control group. Although not statistically significant, the mean numbers of matured (86.8% vs. 85.1%) and fertilized oocytes (84.2% vs. 83.1%), good embryos (62.4% vs. 60.3%), and chemical pregnancy rates (47.2% vs. 46.8%) were slightly higher in the corifollitropin alfa group than in the control group. In contrast, rates of ET cancelled cycles and the OHSS risk were slightly lower in the corifollitropin alfa group (6.2% and 2.7%) than in the control group (8.2% and 3.5%), although these differences were also not statistically significant. CONCLUSION: Although no significant differences were observed, the use of corifollitropin alfa seems to offer some advantages to patients because of its short treatment duration, safety, lower ET cancellation rate and reduced risk of OHSS.

A homolog of splicing factor SF1 is essential for development and is involved in the alternative splicing of pre-mRNA in Arabidopsis thaliana.

During initial spliceosome assembly, SF1 binds to intron branch points and interacts with U2 snRNP auxiliary factor 65 (U2AF65). Here, we present evidence indicating that AtSF1, the Arabidopsis SF1 homolog, interacts with AtU2AF65a and AtU2AF65b, the Arabidopsis U2AF65 homologs. A mutant allele of AtSF1 (At5g51300) that contains a T-DNA insertion conferred pleiotropic developmental defects, including early flowering and abnormal sensitivity to abscisic acid. An AtSF1 promoter-driven GUS reporter assay showed that AtSF1 promoter activity was temporally and spatially altered, and that full AtSF1 promoter activity required a significant proportion of the coding region. DNA chip analyses showed that only a small proportion of the transcriptome was altered by more than twofold in either direction in the AtSF1 mutant. Expression of the mRNAs of many heat shock proteins was more than fourfold higher in the mutant strain; these mRNAs were among those whose expression was increased most in the mutant strain. An RT-PCR assay revealed an altered alternative splicing pattern for heat shock transcription factor HsfA2 (At2g26150) in the mutant; this altered splicing is probably responsible for the increased expression of the target genes induced by HsfA2. Altered alternative splicing patterns were also detected for the transcripts of other genes in the mutant strain. These results suggest that AtSF1 has functional similarities to its yeast and metazoan counterparts.

Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development.

Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.

Post-death cloning of endangered Jeju black cattle (Korean native cattle): fertility and serum chemistry in a cloned bull and cow and their offspring.

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.

The sequence variation responsible for the functional difference between the CONSTANS protein, and the CONSTANS-like (COL) 1 and COL2 proteins, resides mostly in the region encoded by their first exons.

Although the protein CONSTANS (CO) and its close relatives CONSTANS-like (COL) 1 and COL2 exhibit high amino acid sequence similarities, only the CO protein regulates floral induction in Arabidopsis. To investigate the structural basis for the functional differences between CO, COL1, and COL2 in flowering, we performed domain-swapping between CO, COL1, and COL2, and site-directed mutagenesis on the first exon of CO. The results suggest that the lack of flowering promotion activity by COL1 and COL2 is mainly attributed to the differences between CO and the COL1 and COL2 proteins in the amino acid sequence encoded by their first exons.

Effect of Glycosaminoglycans on In vitro Fertilizing Ability and In vitro Developmental Potential of Bovine Embryos.

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 µg/ml of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

Establishment of bovine embryonic stem cell lines using a minimized feeder cell drop.

Bovine embryonic stem cells (ESCs) are a powerful tool for agricultural and biomedical applications. The purpose of this study was to introduce a new method for generating bovine ESCs. Mechanically isolated bovine inner cell masses (ICMs) from in vitro-produced blastocysts were cultured individually on a 10-µL mouse embryonic fibroblast (MEF) feeder cell drop covered with oil. From 126 blastocysts classified by their developmental stage and ICM size, 21 primary bovine ESC-like colonies were formed (16.7%) and established six JNU (Jeju National University)-ibES cell lines (28.6%, 6/21; hatched blastocyst×4, hatching blastocyst×1, and expanded blastocyst×1). These cells exhibited typical ESC morphology, and pluripotency markers were detected through immunocytochemistry, RT-PCR, and real-time RT-PCR, including Oct4, stage-specific embryonic antigen-1 (SSEA-1), Nanog, Tumor rejection antigen-1-81, Rex1, and alkaline phosphatase. Through RT-PCR analysis of spontaneous differentiation, gene expression of all three embryonic germ layers was detected: ectodermal (Pax6 and DBH), mesodermal (CMP and Enolase), and endodermal [alpha fetoprotein (α-FP) and albumin]. In addition, JNU-ibES cell lines were directed differentiated into neuronal (Map2 and Tuj1) and glial (GFAP) cells. Bovine ESC lines had a normal karyotype, with a chromosome count of 58+XY (JNU-ibES-05). This is the first trial investigating a minimized microdrop culture method for the generation of bovine ESCs. These results demonstrated that the minimized MEF feeder cell drop can support the establishment of bovine ESC lines.

Efficient generation of virus-free iPS cells using liposomal magnetofection.

The generation of induced pluripotent stem (iPS) cells is a powerful tool in regenerative medicine, and advances in nanotechnology clearly have great potential to enhance stem cell research. Here, we introduce a liposomal magnetofection (LMF) method for iPS cell generation. Efficient conditions for generating virus-free iPS cells from mouse embryonic fibroblast (MEF) cells were determined through the use of different concentrations of CombiMag nanoparticle-DNA(pCX-OKS-2A and pCX-cMyc)-lipoplexes and either one or two cycles of the LMF procedure. The cells were prepared in a short reprogramming time period (≤ 8 days, 0.032-0.040%). Among the seven LMF-iPS cell lines examined, two were confirmed to be integration-free, and an integration-free LMF-iPS cell line was produced under the least toxic conditions (single LMF cycle with a half-dose of plasmid). This cell line also displayed in vitro/in vivo pluripotency, including teratoma formation and chimeric mouse production. In addition, the safety of CombiMag-DNA lipoplexes for the transfection of MEF cells was confirmed through lactate dehydrogenase activity assay and transmission electron microscopy. These results demonstrated that the LMF method is simple, effective, and safe. LMF may represent a superior technique for the generation of virus-free or integration-free iPS cell lines that could lead to enhanced stem cell therapy in the future.

Identification of marneral synthase, which is critical for growth and development in Arabidopsis.

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3-oxidosqualene (OS), which is cyclized by 2,3-oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.

Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

A meta-analysis of the effects of interleukin-6 -174 G>C genetic polymorphism on acute graft-versus-host disease susceptibility.

BACKGROUND: The interleukin-6 (IL-6) -174 G>C genetic polymorphism has been implicated to play an important role in acute graft-versus-host disease (aGVHD). However, previous studies have yielded inconclusive results as to its role in patient susceptibility to aGVHD, and no study to date has systematically analyzed this polymorphism. OBJECTIVE: A meta-analysis of the published evidence was conducted to estimate the true effect of the IL-6 -174 G>C genetic polymorphism in allogeneic hematopoietic stem cell transplantation (alloHSCT) patients and donors on the risk of aGVHD. METHODS: Seven cohort studies, comprising 1287 recipient and donor pairs, were included after eliminating 62 studies that met the following exclusion criteria: irrelevant studies other than cohort studies, without sufficient data, and with overlapping data. Although interstudy heterogeneity existed, most studies were conducted in the United States or Europe and included adult patients with hematologic disease who received alloHSCT from human leukocyte antigen-matched or identical sibling donors. The effect of the polymorphism on aGVHD risk (grades I-IV, II-IV, and III-IV) was estimated from odds ratios with 95% confidence intervals for the dominant genetic model and recessive model, respectively. RESULTS: Patients who received grafts from donors with the IL-6 G allele experienced more frequent grade I-IV aGVHD (odds ratio = 3.304 [95% confidence interval, 1.456-7.494]) and grade II-IV aGVHD (odds ratio = 1.738 [95% CI, 1.006 - 3.001]). CONCLUSIONS: To our knowledge, this is the first meta-analysis to evaluate the relation between a non-human leukocyte antigen gene polymorphism and the risk of aGVHD. Our meta-analysis combined the results of several studies and demonstrated that the donor IL-6 G allele is associated with an increased risk of grades I-IV and II-IV aGVHD.

The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.

Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.